Quantification of tricyclic glycopeptide in human plasma by UHPLC-MS3 coupled with counter-extraction follow by protein precipitation to enhance sensitivity

Abstract

An ultra-high performance liquid chromatography tandem mass spectrometry cubed (UHPLC/MS³) assay coupled with protein precipitation and counter-extraction for detection of tricyclic glycopeptide vancomycin in human plasma was established and validated in this study. After protein precipitation and counter-extraction with dichloromethane, chromatographic separation of vancomycin and norvancomycin were performed on a reversed phase column (XBridge Peptide BEH C₁₈ column, 2.1 × 100 mm I.D, 3.5 μm). The transition (parent ions → fragment ions → further fragment ions) at m/z 725.3 → 144.1 → 100.1 was used for quantification of vancomycin. The transition (parent ions → fragment ions) at m/z 718.3 → 144.2 was used for detection of norvancomycin. The linear range of the developed analytical method for quantification of vancomycin was 0.5–100 µg/mL (r = 0.9989). The range of intra- and inter-day precisions of the assay among low, medium and high concentrations is between 1.88 % and 6.33 %. The sensitivity of the analytical method was significantly improved by using MS³ technique as monitoring mode and counter-extraction with dichloromethane followed by protein precipitation as sample processing assay. The developed UHPLC/MS³ assay was successfully applied for clinical therapeutic drug monitoring (TDM) of vancomycin in 45 human plasma samples.